Our proposed research has four major goals, which we believe are attainable during the project period. All utilize the synchronous, hour-long differentiation of Naegleria from amebae to flagellates, which involves a dramatic change in cell phenotype, including the morphogenesis of new organelles. The first three goals, closely interrelated, are based on our observation that the tubulin that forms the flagellar microtubules is selectively synthesized de novo during differentiation and is immunochemically distinct from the tubulin that preexists in amebae: 1. Multi-tubulin hypothesis. Determine the nature of the differences between tubulins of Naegleria by continuing our immunochemical study and by selective analysis of the primary structure of tubulins from different cell structures. 2. Regulation of flagellar tubulin synthesis. Isolate and characterize flagellar tubulin mRNA, and determine whether it is synthesized de novo during differentiation. Use purified mRNA to obtain DNA to clone in a plasmid vector. Use the cloned DNA to study the regulation of flagellar tubulin mRNA synthesis. 3. Spatial and temporal regulation of events from synthesis of flagellar tubulin to assembly into flagella. Determine conditions regulating the synthesis of flagellar tubulin. Determine where flagellar tubulin is synthesized, in relation to site of assembly, and whether there is an orderly relationship between synthesis and assembly. 4. Intracellular regulation of cell shape and motility. This goal is based on our discovery of psi, a cell-produced chemical factor that causes shape changes, and which is postulated to regulate intracellular release of calcium ions. Identify psi, study its mode of action, and the "compartmentalization" of psi that occurs during differentiation. Study the morphological and molecular basis of the changes in shape, including especially the role of calcium ions in influencing the assembly and functioning of action-based and tubulin-based systems that determine cell motility and shape.